Bacterial endospores are agents of infectious disease, potential bioweapons, and causes of food spoilage and poisoning. The most notorious among these is Clostridium difficile-associated disease (CDAD) which continues to become more widespread as well are more severe. Elimination of spores from contaminated sites or materials is challenging due to the extreme resistance of these cell types to conventional antimicrobial treatments. Spores that have been triggered to germinate and lose dormancy are much more susceptible to antimicrobial treatments. Activation of a spore's native germination apparatus is a viable strategy for increasing the effectiveness of spore-killing methods. Dormant spores contain highly efficient machinery for sensing favorable changes in their environment and triggering germination. However, even under ideal laboratory germination conditions, up to 5% of a spore population will remain dormant. These "superdormant" spores are resistant to decontamination and are a potential cause of infection at later dates. The proposed research will catalog and quantify the proteomes of the membrane fractions of dormant, germinating, and superdormant spores of the species Bacillus anthracis, Bacillus subtilis, C. difficile, and Clostridium perfringens. Proteome similarities and differences between species will contribute to our understanding of the mechanics of spore germination signaling and progression. Proteome differences between rapidly-germinating and superdormant spores will indicate potential causes of superdormancy. This data will be used to develop models for how the signaling apparatus involved in initiation of germination contributes to superdormancy. Results of the studies will guide further studies on spore germination and the development of more efficient methods of spore decontamination. PUBLIC HEALTH RELEVANCE: Dormant bacterial spores are agents of infectious disease, potential bioweapons, and causes of food spoilage and poisoning. If spores can be caused to germinate, then they are much more susceptible to decontamination treatments. This research will reveal details of the natural germination apparatus, so that better methods for triggering germination can be devised.